INTRODUCTION:

Dolutegravir is chemically designated as Isopropyl (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5] pyrazino[2,1-b][1,3] oxazine-9-carboxamide. Its molecular formula is C20H19F2N3O5, and its molecular weight is 441.37 g/mol

Figure 1. Structure of dolutegravir¹.

Dolutegravir is a HIV-1 integrase inhibitor that blocks the strand transfer step of the integration of the viral genome into the host cell (INSTI). Dolutegravir belongs to a group of HIV drugs called ‘integrase’ inhibitors. Integrase inhibitors block HIV enzyme called integrase. By blocking integrase, it prevent HIV from multiplying and can reduce the amount of HIV in the body. The effect of this drug has no homology in human host cells which gives it an excellent tolerability and minimal toxicity. Dolutegravir was developed by ViiV Healthcare and FDA approved it on August 12, 2013. Dolutegravir is indicated in combination with other antiretroviral agents for the treatment of patients with HIV-1 infection that comply with the characteristics of being adults or children aged 12 years and older and present at least a weight of 40kg². DTG is a drug that is more effective, easier to take and has fewer side effects than alternative drugs that are currently used. Based on new evidence assessing benefits and risks, the WHO recommends the use of the ANTI- HIV drug dolutegravir (DTG) as the preferred first-line and second-line treatment for all populations, including pregnant women and those of childbearing potential³.

In literature review, various methods are available for determination of DTG in drugs by UV^4-5spectroscopy, also DTG in combination with other drug⁶. The present work is therefore focused on to achieve the optimum chromatographic conditions for the determination of DTG in a formulation by UV spectroscopic method, and validate according to ICH guidelines.

MATERIALS AND METHOD:

Materials:

Pharmaceutical grade (>99%) drug was obtained as gift sample from Aadhar Life Sciences Pvt. Ltd, (Solapur, Maharashtra State, India).

Instruments:

Analytical balance (Shimadzu AY220), Sonicator (Microclean-1103), UV-Visible spectrophotometer (Systronic 2201).

Method Development:

Preparation of standard stock solution: Accurately weighed 5mg of Dolutegravir transferred to 10ml volumetric flask. It was dissolved in 5ml of methanol and sonicated for 5 minutes. The volume was made up to mark with distilled water to make up the volume.

Procedure for plotting calibration curve:

For calibration curve in a series of 10ml volumetric flasks, 0. 2 to 1ml of standard solution was pipetted out separately.The volume was completed to the mark using distilled water. The absorbance was measured at wavelength 254nm against blank solution.

Analysis of Dolutegravir in Formulation:

5mg equivalent Dolutegravir tablet was weighed and transferred to the 10ml volumetric flask and dissolved in 5ml of methanol and volume was marked up with distilled water. then it was sonicated for 5min and vortex for 2min. 0.6ml of above solution was pipetted out and transferred to the 10ml volumetric flask and the volume was marked up with distilled water and analysed at 254nm. and % purity of was calculated.

RESULTS AND DISCUSSION:

The absorption spectrum shows λ max of Dolutegravir at 254nm.

Figure 2 UV spectrum of dolutegravir.

The proposed method was validated according to ICH Q28 R1 guidelines for validation of analytical procedure.

Linearity:

Five different concentrations of Dolutegravir were prepared and analysed at wavelength 254nm. The regression coefficient was found to be 0.998. (Table 1).

Table 1 Results of LinearityResults of Linearity

Sr. No.	Concentration(µg/ml)	Absorbance
1	5	0.293
2	10	0.594
3	15	0.912
4	20	1.234
5	25	1.497

Figure 3 Calibration curve for dolutegravir. (Conc.vs.Abs.)

Table 2 Optimization parameters of Dolutegravir

Parameters	Method values
Maximum Wavelength	254nm
Beer’s Law	10-50 µg/ml
Correlation Coefficient (r²)	0.998
Regression Equation	y = 0.030x - 0.008
Slope (m)	0.030
Intercept (c)	0.008

Table 3 Results of Accuracy.

% Conc	Reps	Spiked Conc (ug/ml)	Abs	Amt Recovered (ug/ml)	% Recovery	AVG	STDEV	RSD
80	Rep 1	23.93	0.7290	23.91	99.92	98.17	1.88	1.92
	Rep 2	23.93	0.7017	23.01	96.18
	Rep 3	23.93	0.7181	23.55	98.42
100	Rep 1	29.91	0.9120	29.91	100.00	100.08	0.34	0.34
	Rep 2	29.91	0.9101	29.85	99.79
	Rep 3	29.91	0.9162	30.05	100.46
120	Rep 1	35.89	1.0944	35.89	100.00	100.67	1.72	1.71
	Rep 2	35.89	1.0877	35.67	99.39
	Rep 3	35.89	1.1231	36.83	102.62

Accuracy:

The accuracy was determined by calculating % recoveries of DTG. It was carried out by adding known amounts of each analyte corresponding to three concentration levels 80, 100and 120 of labeled claim. At each level 3 determinations were performed and results were expressed as % recovery. (Table-3).

Range:

Range is an interval between highest and lowest concentration limit of the analyte i.e. 10-50 µg/ml.

Precision:

In instrumental precision were performed at concentration (30µg/ml). The obtained results were found within limit i.e. less than 2%RSD.

Table 4 Results of precision

Sample ID	Abs
Rep 1	0.912
Rep 2	0.9101
Rep 3	0.9162
Rep 4	0.9152
Rep 5	0.9207
AVG	0.91484
STDEV	0.004088
RSD	0.45

Limit of Detection (LOD):

The limit of detection was found to be 0.991 µg/ml.

Limit of Quantification (LOQ):

The limit of quantification was found to be 3.00µg/ml.

Assay:

The assay was performed by using instgra 50mg Tablet at concentration 30µg/ml. The % purity was found to be 101.75 %.

Table 5 Result of assay

Sample ID	Abs	% Assay
Working standard	0.912	-
Drug product	0.928	101.75

CONCLUSION:

An analytical UV spectrophotometric method was developed and validated thoroughly for quantitative determination of Dolutegravir in bulk drug and formulation. The presented method was found to be simple, precise, accurate, rugged, reproducible and gives an acceptable recovery of the analyte, which can be directly easily applied to the analysis of pharmaceutical formulation of Dolutegravir.

ACKNOWLEDGEMENT:

Authors are thankful to the Principal, College of Pharmacy Solapur, for providing the necessary facilities.

REFERENCES:

1. Dolutegravir image [Internet]. Wikipedia. [cited 2020 Aug 10]. Available From: https://en.wikipedia.org/wiki/Dolutegravir

2. Dolutegravir [Internet]. Drug bank. [cited 2020 Aug 10]. Available From: https://www.drugbank.ca/drugs/DB08930

3. Dolutegravir [Internet]. WHO. [cited 2020 Aug 10]. Available From: https://www.who.int/news-room/detail/22-07-2019-who-recommends-dolutegravir-as-preferred-hiv-treatment-option-in-all-populations

4. Bhavar G, Aher K. et al. Development and validation of uv spectrophotometric method for estimation of. Dolutegravir sodium in tablet dosage. Malaysian Journal of Analytical Sciences. 2015; 19(6): 1156 - 1163.

5. K. Bhavyasri et al. Method Development, Validation and Forced Degradation Studies of Dolutegravir, An Anti-Retroviral Drug using Uv-Visible Spectroscopy. Int J Pharm Sci and Scient Res. 2019; 5: (6): 69-74.

6. Shamla Moideen et al. Method Development and Validation of Simultaneous Estimation of Dolutegravir and Lamivudine in Synthetic Mixtures by UV-Visible Spectroscopy. International Journal of Pharmacy and Pharmaceutical Research. 2020; 17 (4): 375-386.

Received on 02.03.2021 Modified on 20.05.2021

Asian J. Pharm. Ana. 2021; 11(3):188-190.

DOI: 10.52711/2231-5675.2021.00032